Addressing a Rule of Thumb: Modeling the Effects of Meteorological Conditions on Prescription of Antimicrobials in Aquaculture

ABSTRACT Meteorological phenomena such as warm summer temperatures and increased numbers of sunlight hours have repeatedly been hypothesized to be associated with infectious diseases and an increased prescription of antimicrobial compounds in Danish aquaculture. To address this hypothesis, we prepared statistical models incorporating aquaculture production data from Danish Aquaculture, detailed records on prescription of antimicrobials from the Danish VETSTAT program, and meteorological data from 2001 to 2019 from the Danish Meteorological Institute. Separate series of models were made and refined for land-based and marine production, respectively. For both production forms, the models identify summer sunlight hours as having a significant influence on antimicrobial use. In addition to summer sunlight, spring sunlight and water temperature were integral, although not statistically significant, parameters when modeling antimicrobial use in marine production. Although the extensive availability and accuracy of relevant data are associated with Danish production, we believe the results allow for more general conclusions on the influence of meteorological parameters on outbreaks of bacterial pathogens in international aquaculture. Such insights could have a substantial impact on prophylactic strategies, fish husbandry, and our understanding of how increasing temperatures may affect future antimicrobial usage in the global aquaculture industry. IMPORTANCE Global aquaculture production has been rapidly increasing for decades and is set to play a pivotal role in feeding a growing human population. Along with the growth in aquaculture production, the annual global use of antimicrobials is estimated to increase by one-third between 2017 and 2030. Using detailed antimicrobial prescription records as a proxy for outbreaks, we were able to evaluate the effects of a variety of meteorological parameters through statistical modeling. Our results lend scientific support to informal observations from the industry, but more importantly, this study provides novel, essential information on the importance of abiotic factors that can, in turn, lead to improved prophylactic efforts and thus help to reduce antimicrobial use in global aquaculture.

General: It would have been interesting to see the AM use in: 1. Marine-based systems 2. Land-based recirculation system 3. Land-based flow through system with ground water supply 4. Land-based system with supply of river/lake water This would pinpoint where any effort should be put to reduce the use of AM. Please consider to add data (in supplement).
In general: Could the authors give a couple of more sentences to compare the use of AM between relevant aquaculture countries. Is the Danish AM use comparable with e.g., Scottish, Faraoe Island, Norwegian countries. I cannot see the mechanistic link between meteorological factors and antimicrobials. The authors require to understand the real environmental conditions in which aquaculture is carried out, the diseases and antibiotic use. The simplistic presentation of the authors of the problem analyzing few meteorological factors seems to have a very low power beyond the statistics to allow to reach relevant and realistic conclusions. In addition, the MS focus on local data (Danish)and is not easy to get a broader analysis on the subject, do not use the existing knowledge in other countries and does not use the existing international literature on antimicrobial use and aquaculture development.

Reviewer #3 (Comments for the Author):
Overall this is an exceptionally well written paper, addressing a a clear and interesting question, conducted to a very high quality. There are no major issues that need addressing, but I have a few thoughts: 1. Abstract L15-16. Please can you be more specific and precise about the two main results (the factors in the best fit models for marine and land systems) in your description of the results.
2. Abstract: Some readers would also like to see quantification in the abstract (e.g. p values or AIC values); I make students do it but am personally less bothered in 'actual' manuscript abstracts; I leave the choice to the discretion of the editor.
3. Figures 2 and 4. While I appreciate the creativity in showing these data in this way, these figures are somewhat unusual. Have the authors considered a more 'standard' approach, that is, to display the correlations as a correlation matrix, coloured accordingly? (I would normally cluster the matrix too so that correlated variables appear close to each other, but not show any underlying trees, as the purpose of the clustering would be cosmetic). I would find such an approach easier to parse, but that is a matter of taste rather than correctness. I would suggest that the authors produce alternative figures as I have suggested, and then see which they prefer -if that is what they have then fine. 4. L251-252. Please be more precise in describing the Land3 model in the text of the Results so it can be fully understood without reference to the table.
Staff Comments:

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Response to reviewers
Please find our point-by-point responses below. The following contains the review comments by all three reviewers, with our responses included in italics. For a detailed overview of all changes made, please see the submitted mark-up .docx-file.
Reviewer #1 (Comments for the Author): This manuscript described any relationship between environmental temperature and the use of antimicrobials (AM). To be honest I am not qualified to evaluate the mathematical modeling and the statistical methods used in this study. But, I have reviewed the other parts. Please consider the following points in the revision: Line 218: Please check the AM use. The authors sate gram per kilo weight. Do you mean body weight. It appears that the AM use is thus quite excessive compared with e.g., Norwegian aquaculture.
-This was an error on our side. The absolute annual AM use is in kg, and the relative AM use is in gram/ton. Not gram/kg as previously written. This has now been corrected throughout the manuscript.
General: It would have been interesting to see the AM use in: 1. Marine-based systems 2. Land-based recirculation system 3. Land-based flow through system with ground water supply 4. Land-based system with supply of river/lake water This would pinpoint where any effort should be put to reduce the use of AM. Please consider to add data (in supplement).
-Absolutely, however we are limited by the way that AM use is reported. While the collection of AM data is very thorough, this is the partitioning of data that we had to work with.
In general: Could the authors give a couple of more sentences to compare the use of AM between relevant aquaculture countries. Is the Danish AM use comparable with e.g., Scottish, Faraoe Island, Norwegian countries.
-This is a good point. For context, AM use from Norwegian and UK aquaculture has now been introduced in the discussion, as follows (Lines 373-380 in the revised marked up manuscript): "Antimicrobial use varies considerably worldwide (1). Looking to major European aquaculture producing countries for context, Norwegian aquaculture has had an annual total AM use of 641 kg from 2010-2019, despite a considerably higher annual production, while AM use from the United Kingdom were a total of 160 kg in trout production and 5600 kg in salmon production in 2020 (2,3). In 2019 the relative AM use for Norway was 0.15 mg per population correction unit (kg live-weight slaughtered biomass) across all aquaculture production, while in the United Kingdom it was 13 mg/kg and 10 mg/kg, for salmon and trout production respectively. Beyond Europe, Schar et al estimate a worldwide mean use of 103 mg active compound per kg trout produced."

tons), B) absolute AM use (kg) and C) relative AM use (g/ton) in land-based (green) and marine (blue) production."
I feel that Figure 5 for me ok (-), but too speculative for many readers, to be in the main text. Please consider to have this figure as supplement, as there are uncertainties how much each factors contributes to AM use.
-We appreciate this suggestion. However, as evident from comments made during this review, we feel that it is very important to note the influences of stressors and production environment parameters that are not considered in this study. We therefore consider this figure quite important to the discussion of the results that we present and prefer to keep the figure in the main text. This suggestion does raise an important point about the relative impacts of each factor. To avoid any confusion regarding the arbitrary sizes of the figure components, the legend has now been changed to the following: " Figure 5: A graphical representation of factors expected to affect AM use. The fluctuation in AM usage that lies at the center of this study, is likely affected by multiple factors. These include vaccine use and -efficacy, stocking densities, feeding programs, as well as various external stressors, such as handling stress and environmental stressors, that are beyond the scope of this study. The size of each ellipsoid is arbitrary, and thus does not reflect relative suspected impacts." Reviewer #2 (Comments for the Author): I cannot see the mechanistic link between meteorological factors and antimicrobials. The authors require to understand the real environmental conditions in which aquaculture is carried out, the diseases and antibiotic use. The simplistic presentation of the authors of the problem analyzing few meteorological factors seems to have a very low power beyond the statistics to allow to reach relevant and realistic conclusions. In addition, the MS focus on local data (Danish)and is not easy to get a broader analysis on the subject, do not use the existing knowledge in other countries and does not use the existing international literature on antimicrobial use and aquaculture development.
-We respectfully disagree with reviewer 2. We acknowledge that the aquaculture production, like any other production form, is highly complex and that no single factor can explain variations in eg. AM use. For that exact reason, we have been careful not to claim that the mechanisms described in this manuscript are the sole reasons for the observed fluctuations. We simply wanted to investigate if there were patterns in historic meteorological data that correlated with AM usage, and if any such patterns could plausibly have an effect on treatment-requiring outbreaks of bacterial pathogens. We believe that we have clearly stated that any weather-related mechanisms will be part of a larger interplay between several other stressors, prophylactic strategies and environmental conditions, as shown in figure 5. As requested by reviewers 1 and 2, contextual data regarding AM use in other countries have been included in the manuscript.
Reviewer #3 (Comments for the Author): Overall this is an exceptionally well written paper, addressing a a clear and interesting question, conducted to a very high quality. There are no major issues that need addressing, but I have a few thoughts: -We appreciate the effort and the comments made.
1. Abstract L15-16. Please can you be more specific and precise about the two main results (the factors in the best fit models for marine and land systems) in your description of the results.
-Yes we can. Without introducing any P-or AIC-values (see point 2 below), we have changed the sentence to the following (lines 15-19 in the revised manuscript): "Separate series of models were made and refined for land-based and marine production, respectively. For both production forms the models identify summer sunlight hours as having a significant influence on antimicrobial use. In addition to summer sunlight, spring sunlight and water temperature were integral, although not statistically significant parameters when modelling antimicrobial use in the marine production." Hopefully this has improved the specificity sufficiently. Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.
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